LOCUS       sequence-441065-        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of iGeoCas9(C2) construct with EGFP-targeting 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  AUTHORS   Eggers AR, Chen K, Soczek KM, Tuck OT, Doherty EE, Xu B, Trinidad 
            MI, Thornton BW, Yoon PH, Doudna JA
  TITLE     Rapid DNA unwinding accelerates genome editing by engineered 
            CRISPR-Cas9.
  JOURNAL   Cell. 2024 May 13:S0092-8674(24)00457-4. doi: 
            10.1016/j.cell.2024.04.031.
  PUBMED    38781968
REFERENCE   2  (bases 1 to 8308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2024 
            May 13:S0092-8674(24)00457-4. doi: 10.1016/j.cell.2024.04.031."
COMMENT     Sequence Label: HuEx-2xNLS-iGeoCas9(C2)-2xNLS_EGFP-g1
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        657..905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     657..677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     828..847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1087..1372
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1374..1651
                     /label=chicken beta-actin promoter
     intron          1652..1879
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1907..1916
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1919..1939
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1949..1969
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5252..5272
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5282..5302
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5312..5368
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5369..5965
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5862..5882
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5999..6016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6005..6212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6221..6361
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6221..6350
                     /label=AAV2 ITR
     rep_origin      6436..6891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6523..6542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6733..6754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6908..6927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7027..7049
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7087..7105)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7173..7277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7278..7346,7347..8138)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7496..7515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt actagtgagg
      661 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      721 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      781 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      841 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      901 acaccgcacg acttcttcaa gtccgccagt catagttccc ctgagaaatc agggttacta
      961 tgataagggc tttctgccta aggcagactg acccgcggcg ttggggatcg cctgtcgccc
     1021 gcttttggcg ggcattcccc atcctttttt ttaaggttgg gccgctcgag ctagtatagc
     1081 ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     1201 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     1261 tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg
     1321 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
     1381 gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta
     1441 tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc
     1501 gccggggggg gggggggggg gsgrggggsg gggsggggsg rggcggagag gtgcggcggc
     1561 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     1621 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
     1681 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
     1741 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta
     1801 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc
     1861 tgaaatcact ttttttcagg ttggaccggt gccacctcga gtcgacgcca ccatgggtcc
     1921 taagaaaaag aggaaggtgg gaggctcacc aaagaagaaa agaaaggtag gcggctctaa
     1981 cgccatgaga tacaaaatcg gcctggacat tggcatcacc agcgtgggct gggccgtgat
     2041 gaacctggat atccctagaa ttgaggacct gggcgtgaga atctttgatc gggctgaaaa
     2101 ccctcagacg ggcgaaagcc tggctctgcc aagaagactg gcaagatccg ccagacggag
     2161 actccggaga agaaagcaca gattggagag aatcagaaga ctggtgatca gagaaggcat
     2221 cctgacaaag gaggaactgg acaagctgtt tgaagagaaa cacgagatcg acgtgtggca
     2281 gctgagggtg gaagccctgg acagaaagct gaacaacgac gagctggccc gagtgctgtt
     2341 acacctggcc aaacggcggg gattcaagtc taacaggaaa agcgagagaa gcaacaaaga
     2401 gaactctaca atgctgaagc acatcgaggg aaacagagcc atcctgagct cttacagaac
     2461 agtgggcgag atgatcgtga aagatcctaa gttcgccctg cataagcgga acaaaggcga
     2521 aaactacatc aacaccatcg ccagagatga cctggagagg gagatcagac tgatcttctc
     2581 caaacagagg gagttcggcg acatgagctg caccgaagag ttcgagaacg agtacatcac
     2641 aatctgggct agccagcggc ctgtggcctc caaggacgac atcgaaaaga aggttggctt
     2701 ctgcacattc gagcctaagg aaaaaagagc tcctaaggcc acatatacct tccagagctt
     2761 tatcgcctgg gaacacatca ataagctgcg gctgatctct ccaagtggcg cccggggcct
     2821 caccgacgag gaacgtagac tgctgtacga gcaggccttt cagaagaaca agatcaccta
     2881 ccacgatatc cgtacactgc tgcacctgcc cgatgataca tacttcaagg gaatcgtgta
     2941 cgaccgcggt gagagccgga agcagaacga gaacatccgg ttcctggagc tcgacgccta
     3001 tcaccagatc cggaaggccg tggacaaggt ttacggcaag ggcaagagca gctcttttct
     3061 gcctatcgac ttcgacacct tcggctacgc cctgaccctg tttaaggacg atgccgacat
     3121 ccactcttac ctgcgcaacg agtacgagca aaatggcaag cggatgccta atctggctaa
     3181 caaagtgtat gacaacgagc tgatcgagga acttctgaac ctgagcttca caaagttcgg
     3241 gcatctgtct ctgaaggccc tgcggtctat cctgccgtac atggaacaag gcgaagtgta
     3301 cagctctgcc tgtgaacggg ccggctacac cttcaccggc cccaaaaaaa agcagaagac
     3361 catgctgctg cctaacatcc agcctattgc caacccggtg gtcatgagag ccctgactca
     3421 ggctagaaag gtggtgaacg ccatcatcaa gaagtacggc agccctgtga gcatccacat
     3481 cgaactggct agagacctgt cccagacctt cgacgaaaga cggaagacca agaaagagca
     3541 ggatgagaac agaaagaaga atgaaaccgc cattcggcaa ctgatggaat acggcctgac
     3601 actgaatccc accggtcatg acatcgtgaa gttcaagctg tggagcgaac agaacggcag
     3661 atgcgcctac agcctgcagc ccatcgagat cgaacggctg ctggagcctg gctacgtgga
     3721 ggtggatcac gtgattcctt acagcagaag cctggatgac agctacacca acaaggtgct
     3781 ggtgctgacc agagagaata gagagaaggg caacagaatc cctgccgagt acctgggagt
     3841 gggcacagag agatggcagc agttcgagac atttgtgctg accaacaagc agttcagcaa
     3901 gaagaaacgg gaccggctgc tgagactgca ctacgacgag aacgaggaaa ccgagttcaa
     3961 gaacagaaac ctgaacgaca ccagatacat cagcagattc ttcgccaact tcatcagaga
     4021 gcacctgaag ttcgccgagt ctgacgacaa gcaaaaagtg tacacagtga atggaagagt
     4081 gacagcccac ctcagaagcc ggtgggagtt caacaaaaat agagaggaga gcgacctgca
     4141 ccacgccgtt gacgccgtga tcgtggcttg taccacccct agtgacatcg ccaaggtgac
     4201 agccttctac cagagaagag aacagaacaa ggaactggcc aagaaaacag aaccccactt
     4261 ccctcagcct tggcctcact ttgccgacga gctaagagcc cggttgagca agcatcctaa
     4321 agagagcata aaagctttaa atctgggcaa ctacgatgat cagaaactgg aatccctgca
     4381 accagtcttt gtgtccagaa tgcctaaacg cagcgtgacc ggcgccgctc acagagaaac
     4441 tctgagaaga tacgtgggca tcgatgagcg tagcggcaag attcagaccg tggtgaaaac
     4501 caagcttagc aaaatcaagc tggacgccag cggacacttc cccatgtacg gcaaagagag
     4561 cgatccccgc acatatgagg ccatccgaca gagactgctg gaacacaaca acgaccctaa
     4621 gaaggcattc cagggccctc tgtacaagcc aaagaagaac ggcgagcctg gccccgttat
     4681 acggaccgtc aagatcatcg ataccagaaa ccaggtaatc cctctgaatg atggcaagac
     4741 cgtggcctac aacagcaata tcgtgagagt ggacgtgttc gaaaaagacg gaaagtatta
     4801 ctgcgtgcca gtgtacacca tggacatcat gaagggcatc ctgccaaaca aggcaatcga
     4861 gcccaacaag ccctattctg aatggaagga gatgaccgag gactatacct tcagattcag
     4921 cctgtacccc aatgatctga tccggatcga actgcctaga gagaagaccg tcaagacagc
     4981 cgccggcgaa gaaataaacg tgaaggacgt gttcgtgtac tacaagacaa tcgatagcgc
     5041 caatggaggc ctcgaactga tcagccacga ccacagattt agcctgagag gcgtcggcag
     5101 ccggaccctg aagagattcg agaaatacca ggtggacgtg ctgggaaata tctacaaggt
     5161 gagaggcgag aaaagagttg gactggcttc tagcgcccac agcaaacccg gcaagaccat
     5221 cagacctctg cagagcaccc gggatggcag ccccaaaaaa aagcggaagg tgggcggaag
     5281 ccctaagaag aagagaaaag tgggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     5341 cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac
     5401 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     5461 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     5521 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     5581 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     5641 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     5701 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     5761 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     5821 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     5881 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     5941 catgacccgc aagcccggtg cctgaacgcg ttaagaattc ctagagctcg ctgatcagcc
     6001 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     6061 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     6121 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     6181 gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag
     6241 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     6301 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     6361 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     6421 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6481 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     6541 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6601 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     6661 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6721 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     6781 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     6841 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     6901 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     6961 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     7021 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     7081 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     7141 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     7201 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     7261 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     7321 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     7381 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     7441 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     7501 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     7561 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     7621 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     7681 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     7741 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     7801 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     7861 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7921 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     7981 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     8041 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     8101 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     8161 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     8221 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     8281 accccgtaga aaagatcaaa ggatcttc
//
